Amino-methyl substituted tetracycline compounds

ABSTRACT

Aminomethyl substituted tetracycline compounds, pharmaceutical compositions, and methods of use thereof are discussed.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 10/737,361, filed Dec. 15, 2003, allowed, which isa continuation of U.S. patent application Ser. No. 10/384,855, filedMar. 10, 2003, abandoned, and claims priority to U.S. Provisional PatentApplication Ser. No. 60/395,495, filed on Jul. 12, 2002 (expired); andU.S. Provisional Patent Application Ser. No. 60/362,654, filed Mar. 8,2002 (expired). Each of the aforementioned applications are herebyincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The development of the tetracycline antibiotics was the direct result ofa systematic screening of soil specimens collected from many parts ofthe world for evidence of microorganisms capable of producingbacteriocidal and/or bacteriostatic compositions. The first of thesenovel compounds was introduced in 1948 under the name chlortetracycline.Two years later, oxytetracycline became available. The elucidation ofthe chemical structure of these compounds confirmed their similarity andfurnished the analytical basis for the production of a third member ofthis group in 1952, tetracycline. A new family of tetracyclinecompounds, without the ring-attached methyl group present in earliertetracyclines, was prepared in 1957 and became publicly available in1967.

Recently, research efforts have focused on developing new tetracyclineantibiotic compositions effective under varying therapeutic conditionsand routes of administration. New tetracycline analogues have also beeninvestigated which may prove to be equal to or more effective than theoriginally introduced tetracycline compounds. Examples include U.S. Pat.Nos. 3,957,980; 3,674,859; 2,980,584; 2,990,331; 3,062,717; 3,557,280;4,018,889; 4,024,272; 4,126,680; 3,454,697; and 3,165,531. These patentsare representative of the range of pharmaceutically active tetracyclineand tetracycline analogue compositions.

Historically, soon after their initial development and introduction, thetetracyclines were found to be highly effective pharmacologicallyagainst rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, and psittacosis. Hence, tetracyclines becameknown as “broad spectrum” antibiotics. With the subsequent establishmentof their in vitro antimicrobial activity, effectiveness in experimentalinfections, and pharmacological properties, the tetracyclines as a classrapidly became widely used for therapeutic purposes. However, thiswidespread use of tetracyclines for both major and minor illnesses anddiseases led directly to the emergence of resistance to theseantibiotics even among highly susceptible bacterial species bothcommensal and pathogenic (e.g., pneumococci and Salmonella). The rise oftetracycline-resistant organisms has resulted in a general decline inuse of tetracyclines and tetracycline analogue compositions asantibiotics of choice.

SUMMARY OF THE INVENTION

In one embodiment, the invention pertains to tetracycline compounds ofthe formula (I):

wherein

R¹ and R² are linked to form a ring, or pharmaceutically acceptablesalts, prodrugs and esters thereof.

The invention also pertains, at least in part, tetracycline compounds ofthe formula:

pharmaceutically acceptable salts, esters or prodrugs thereof.

The invention also pertains, at least in part, to tetracycline compoundsof the formula (II):

wherein:

J¹ and J² are each independently hydrogen, aryl, sulfonyl, acyl, orlinked to form a ring, provided that at least one of J¹ or J² is nothydrogen;

J³ and J⁴ are each alkyl, halogen, or hydrogen;

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,arylalkynyl, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(=Z′)ZR^(9a);

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f)

R^(9a), R^(9b), R^(9c), R^(9d), and R^(9e) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts, esters, and prodrugs thereof.

The invention also pertains, at least in part, to tetracycline compoundsof formula (III):

wherein:

J⁵ and J⁶ are each independently hydrogen, alkyl, alkenyl, alkynyl,aryl, sulfonyl, acyl, alkoxycarbonyl, alkaminocarbonyl,alkaminothiocarbonyl, substituted thiocarbonyl, substituted carbonyl,alkoxythiocarbonyl, or linked to form a ring;

J⁷ and J⁸ are each alkyl, halogen, or hydrogen;

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

The invention also pertains to compounds of the formulae shown in Table1 and pharmaceutically acceptable esters, salts, and prodrugs thereof.

The invention also pertains, at least in part, to pharmaceuticalcompositions comprising aminomethyl tetracycline compounds of formulae(I), (II), (III), Table 1, or otherwise described herein. Thepharmaceutical compositions preferably comprise an effective amount of aminocycline compound and a pharmaceutically acceptable carrier.

In another embodiment, the invention also pertains, at least in part, tomethods of using the aminomethyl tetracycline compounds of the invention(e.g., of formula (I), (III), (III), Table 1, or otherwise describedherein), to treat tetracycline associated states in subjects.

In an embodiment, the invention pertains, at least in part, to a methodfor the synthesis of an aminoalkyl tetracycline compound. The methodincludes contacting a tetracycline compound with an aminoalkylatingreagent under appropriate conditions, such that an aminoalkyltetracycline compound is formed.

In another embodiment, the invention pertains to pharmaceuticalcompositions containing the aminoalkyltetracycline compounds of theinvention and aminoalkyltetracycline compounds synthesized by themethods of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In an embodiment, the invention pertains to tetracycline compounds ofthe formula (I):

wherein

R¹ and R² are linked to form a ring, or pharmaceutically acceptablesalts, prodrugs and esters thereof.

In one embodiment, R¹ and R² are linked to form a five or six memberedring. In another, R¹ and R² are linked to form a six membered ring. R¹and R² may be linked by a chain of atoms such as, for example,—(CH₂)₅₋₆, —(CH₂)₁₋₅—CH═CH—(CH₂)₁₋₅—, —(CH₂)₁₋₅—O—(CH₂)₁₋₅—,—(CH₂)₁₋₅—NR—(CH₂)₁₋₅, etc. The ring formed may be saturated orunsaturated. For example, R¹ and R² may be linked to form a piperidinering, morpholine ring, pyridine ring, or a pyrazinyl ring.

In a further embodiment, the tetracycline compound is:

pharmaceutically acceptable salts, esters or prodrugs thereof.

In one embodiment, the invention pertains to aminomethyl tetracyclinecompounds of the formula (II):

wherein:

J¹ and J² are each independently hydrogen, aryl, sulfonyl, acyl, orlinked to form a ring, provided that at least one of J¹ or J² is nothydrogen;

J³ and J⁴ are each alkyl, halogen, or hydrogen;

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,arylalkynyl, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(=Z′)ZR^(9a);

Z is CR^(9d)R^(9e), S, NR^(9b) or O;

Z′ is O, S, or NR^(9f);

R^(9a), R^(9b), R^(9c), R^(9d), and R^(9e) are each independentlyhydrogen, acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic,heteroaromatic or a prodrug moiety;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts, esters, and prodrugs thereof.

In an embodiment, the tetracycline compound is a oxytetracyclinecompound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are eachmethyl, R⁵ is OH, X is CR⁶R^(6′), R⁶ is OH, and R^(6′) is CH₃). Inanother embodiment, the aminoalkyl tetracycline compound is ademeclocycline compound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) andR^(4″) are each methyl, R⁵ is hydrogen, X is CR⁶R^(6′), R⁶ is OH, R^(6′)is hydrogen, and R⁷ is chlorine). In another embodiment, the aminoalkyltetracycline compound is a methacycline compound (e.g., wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are each methyl, R⁵ is OH, X isCR⁶R^(6′), R⁶ and R^(6′) are, taken together, CH₂). In anotherembodiment, the aminoalkyl tetracycline compound is a doxycyclinecompound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are eachmethyl, R⁵ is OH, X is CR⁶R^(6′), R⁶ is OH, and R^(6′) is CH₃). Inanother embodiment, the aminoalkyl tetracycline compound is achlorotetracycline compound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′)and R^(4″) are each methyl, R⁵ is hydrogen, X is CR⁶R^(6′), R⁶ is OH,R^(6′) is CH₃, and R^(4″) is chlorine). In another embodiment, theaminoalkyl tetracycline compound is a tetracycline compound (e.g.,wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are each methyl, R⁵ ishydrogen, X is CR⁶R^(6′), R⁶ and R^(6′) are each hydrogen and R⁷ isN(CH₃)₂). In a further embodiment, R⁵ of formula I is a protectedhydroxyl group, e.g. a prodrug moiety. Examples of prodrug moietiesinclude, for example, acyl esters and esters. In certain embodiments,the prodrug moiety is aroyl, alkanoyl, or alkaroyl and may or may not becleaved in vivo to the hydroxyl group. In certain embodiments, R⁴ ishydrogen.

In an embodiment, J³ and J⁴ are hydrogen. In another embodiment, J¹ maybe substituted or unsubstituted alkyl. J¹ also may be sulfonyl or J¹ andJ² may be linked to form a ring. In a further embodiment, J¹ maybeheteroaryl or substituted carbonyl.

Examples of aminoalkyltetracycline compounds synthesized by methods ofthe invention include, but are not limited to, compounds of thefollowing formulae:

wherein

R is substituted or unsubstituted alkyl, alkenyl, alkynyl, halogen,alkoxy; and

Y is N, O, or S, or pharmaceutically acceptable salts or prodrugsthereof.

In another embodiment, the aminoalkyl tetracycline compound of theinvention may be a compound of the formula (II):

wherein:

J⁵ and J⁶ are each independently hydrogen, alkyl, alkenyl, alkynyl,aryl, sulfonyl, acyl, alkoxycarbonyl, alkaminocarbonyl,alkaminothiocarbonyl, substituted thiocarbonyl, substituted carbonyl,alkoxythiocarbonyl, or linked to form a ring;

J⁷ and J⁸ are each alkyl, halogen, or hydrogen;

X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O;

R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety;

R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen,or hydrogen;

R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety;

R⁵ is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy;

R⁶ and R^(6′) are each independently hydrogen, methylene, absent,hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;

R⁷ is hydrogen;

R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl;

R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl; and

Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.

In an embodiment, the tetracycline compound is a oxytetracyclinecompound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are eachmethyl, R⁵ is OH, X is CR⁶R^(6′), R⁶ is OH, and R^(6′) is CH₃). Inanother embodiment, the aminoalkyl tetracycline compound is ademeclocycline compound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) andR^(4″) are each methyl, R⁵ is hydrogen, X is CR⁶R^(6′), R⁶ is OH, R^(6′)is hydrogen, and R⁷ is chlorine). In another embodiment, the aminoalkyltetracycline compound is a methacycline compound (e.g., wherein R⁴ isNR^(4′)R^(4″), R^(4′) and R^(4″) are each methyl, R⁵ is OH, X isCR⁶R^(6′), R⁶ and R^(6′) are, taken together, CH₂). In anotherembodiment, the aminoalkyl tetracycline compound is a doxycyclinecompound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are eachmethyl, R⁵ is OH, X is CR⁶R^(6′), R⁶ is OH, and R^(6′) is CH₃). Inanother embodiment, the aminoalkyl tetracycline compound is achlorotetracycline compound (e.g., wherein R⁴ is NR^(4′)R^(4″), R^(4′)and R^(4″) are each methyl, R⁵ is hydrogen, X is CR⁶R^(6′), R⁶ is OH,R^(6′) is CH₃, and R⁷ is chlorine). In another embodiment, theaminoalkyl tetracycline compound is a tetracycline compound (e.g.,wherein R⁴ is NR^(4′)R^(4″), R^(4′) and R^(4″) are each methyl, R⁵ ishydrogen, X is CR⁶R^(6′), R⁶ and R^(6′) are each hydrogen and R⁷ isN(CH₃)₂). In a further embodiment, R⁵ of formula I is a protectedhydroxyl group, e.g. a prodrug moiety. Examples of prodrug moietiesinclude, for example, acyl esters and esters. In certain embodiments,the prodrug moiety is aroyl, alkanoyl, or alkaroyl and may or may not becleaved in vivo to the hydroxyl group. In certain embodiments, R⁴ ishydrogen.

In an embodiment, J⁷ and J⁸ are hydrogen. In another embodiment, J⁵ maybe substituted or unsubstituted alkyl. J⁵ also may be sulfonyl or J⁵ andJ⁶ may be linked to form a ring. In a further embodiment, J⁵ maybeheteroaryl or substituted carbonyl.

Examples of aminoalkyltetracycline compounds synthesized by methods ofthe invention include, but are not limited to, compounds of thefollowing formulae:

wherein

R is substituted or unsubstituted alkyl, alkenyl, alkynyl, halogen,alkoxy; and

Y is N, O, or S, or pharmaceutically acceptable salts or prodrugsthereof.

Other aminoalkyl tetracycline compounds of the invention are shown inTable 1. The invention includes pharmaceutically acceptable esters,salts, and prodrugs of the compounds shown in Table 1. TABLE 1

The invention also pertains, at least in part to a method for thesynthesis of aminoalkyl tetracycline compounds, such as those describedabove. The method includes contacting a tetracycline compound with anaminoalkylating reagent under appropriate conditions to form anaminoalkyl tetracycline compound.

The term “tetracycline compound” includes many compounds with a similarring structure to tetracycline. Examples of tetracycline compoundsinclude: tetracycline, chlortetracycline, oxytetracycline,demeclocycline, methacycline, sancycline, doxycycline, minocycline,chelocardin, rolitetracycline, lymecycline, apicycline; clomocycline,guamecycline, meglucycline, mepylcycline, penimepicycline, pipacycline,etamocycline, penimocycline, etc. Other derivatives and analoguescomprising a similar four ring structure are also included (for areview, see W. Rogalski, “Chemical Modifications of Tetracyclines,” theentire contents of which are hereby incorporated herein by reference).Table 2 depicts tetracycline and several known other tetracyclinederivatives. TABLE 2

Other tetracycline compounds which may be modified using the methods ofthe invention include, but are not limited to,6-demethyl-6-deoxy-4-dedimethylaminotetracycline; tetracyclino-pyrazole;7-chloro-4-dedimethylaminotetracycline;4-hydroxy-4-dedimethylaminotetracycline;12α-deoxy-4-dedimethylaminotetracycline;5-hydroxy-6α-deoxy-4-dedimethylaminotetracycline;4-dedimethylamino-12α-deoxyanhydrotetracycline;7-dimethylamino-6-demethyl-6-deoxy-4-dedimethylaminotetracycline;tetracyclinonitrile; 4-oxo-4-dedimethylaminotetracycline 4,6-hemiketal;4-oxo-11a Ch1-4-dedimethylaminotetracycline-4,6-hemiketal;5a,6-anhydro-4-hydrazono-4-dedimethylamino tetracycline;4-hydroxyimino-4-dedimethylamino tetracyclines;4-hydroxyimino-4-dedimethylamino 5a,6-anhydrotetracyclines;4-amino-4-dedimethylamino-5a,6 anhydrotetracycline;4-methylamino-4-dedimethylamino tetracycline;4-hydrazono-11a-chloro-6-deoxy-6-demethyl-6-methylene-4-dedimethylaminotetracycline; tetracycline quaternary ammonium compounds;anhydrotetracycline betaines; 4-hydroxy-6-methyl pretetramides; 4-ketotetracyclines; 5-keto tetracyclines; 5a,11a dehydro tetracyclines; 11aC1-6, 12 hemiketal tetracyclines; 11a C1-6-methylene tetracyclines; 6,13diol tetracyclines; 6-benzylthiomethylene tetracyclines;7,11a-dichloro-6-fluoro-methyl-6-deoxy tetracyclines; 6-fluoro(α)-6-demethyl-6-deoxy tetracyclines; 6-fluoro (β)-6-demethyl-6-deoxytetracyclines; 6-α acetoxy-6-demethyl tetracyclines; 6-βacetoxy-6-demethyl tetracyclines; 7,13-epithiotetracyclines;oxytetracyclines; pyrazolotetracyclines; 11a halogens of tetracyclines;12a formyl and other esters of tetracyclines; 5,12a esters oftetracyclines; 10,12a-diesters of tetracyclines; isotetracycline;12-a-deoxyanhydro tetracyclines;6-demethyl-12a-deoxy-7-chloroanhydrotetracyclines; B-nortetracyclines;7-methoxy-6-demethyl-6-deoxytetracyclines;6-demethyl-6-deoxy-5a-epitetracyclines; 8-hydroxy-6-demethyl-6-deoxytetracyclines; monardene; chromocycline; 5a methyl-6-demethyl-6-deoxytetracyclines; 6-oxa tetracyclines, and 6 thia tetracyclines.

The term “aminoalkyl tetracycline compounds” includes tetracyclinecompounds with an aminoalkyl substituent, (e.g., —CH₂NR′R″) at the 7and/or 9 positions. In an embodiment, the substitution at the 7 and/or 9position enhances the ability of the tetracycline compound to performits intended function (e.g., as an antibiotic, to treat a tetracyclinecompound responsive state, etc.). The term “aminoalkylating reagent”includes reagents which are capable of contacting the tetracyclinecompound and, under appropriate conditions, reacting with it to form anaminoalkyl tetracycline compound. The amino alkylating reagent may beadded to the reaction mixture or may be formed in situ. Examples ofaminoalkylating reagents, include, but are not limited to, compounds ofthe formula (IV):

wherein

-   -   R^(a′) and R^(a″) are each independently hydrogen or halogen;    -   R′ is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, or        halogen;

and

-   -   R″ is hydrogen or optionally linked to R′ to form a 4-8 membered        ring.        The ring may be optionally substituted, e.g., with halogens and        may comprise carbons and/or heteroatoms such as oxygen,        nitrogen, and sulfur. R′ may be further substituted with any        substituent which does not prevent the reagent from reacting        with the tetracycline compound of the invention, under the        appropriate conditions. In another further embodiment, R′ is        alkyl, e.g., unsubstituted or substituted (e.g., with halogens,        e.g., chlorine, fluorine, bromine, iodine, etc.). In another        embodiment, R′ is aryl, e.g., phenyl, e.g., unsubstituted or        substituted (e.g., with halogens (e.g., chlorine, bromine,        fluorine, etc.), hydroxy, alkoxy, esters, amino, etc.). In        another embodiment, R^(a′) and R^(a″) are each hydrogen. Other        examples of aminoalkylating reagents include        N-hydroxymethylphthalimide.

Examples of amino-alkylating reagents include, but are not limited to:

The term “appropriate conditions” include those conditions under whichthe aminoalkylating reagent and the tetracycline compound interact suchthat an aminoalkyl tetracycline compound is formed. In an embodiment,the appropriate conditions comprise treating the tetracycline compoundwith an acid prior to, or concurrently with the addition of theaminoalkylating reagent to the reaction mixture. Examples of acids whichmaybe used alone or in combination include acids known in the art, aswell as, sulfuric acid, hydrofluoric acid (HF), methanesulfonic acid,trifluoromethane sulfonic acid, hydrochloric acid, hydrochloric acid inaqueous ethanol, acetic acid, methanesulfonic acid, and trifluoroaceticacid (TFA). In a further embodiment, appropriate conditions may alsocomprise treating the resulting tetracycline compound with a reactionquenching agent (e.g., water).

Each of the reactions described below may be applied to othertetracycline compounds described above. In addition, although many ofthe schemes depicts substituting the tetracycline compound at the 9position, similar substituents can be added at the 7 position by using aprotecting group at the 9 position, (e.g., such as t-butyl).

Scheme 1 depicts the reaction of sancycline with an aminoalkylatingreagent under appropriate conditions such that an aminoalkyltetracycline compound is formed.

Scheme 2 shows two aminoalkylations of a tetracycline compound withaminoalkylating reagents which comprise a 5 membered ring. Similarreactions can be also be carried out using reagents, with, for example,6- or 7-membered rings.

As shown in Scheme 3 below, the synthesis of 7-monosubstitutedaminomethyl tetracyclines may be synthesized using protecting groups(i.e. the 9-t-butyl protecting group) to be cleaved using art recognizedtechniques, such as acid. Examples of acids which can be used include,but are not limited to, HF, trifluoroacetic acid (TFA), H₂SO₄ andmixtures thereof. In this way, regioselective aminomethylation atposition 7 is achieved.

In a further embodiment, the appropriate conditions may further comprisetreating the reaction mixture (which may comprise an intermediateaminoalkyl tetracycline compound) with a derivatizing agent undersecondary appropriate conditions such that the desired aminoalkyltetracycline compound is formed. The reactions in Scheme 4 are shown forthe 9 position, but the reactions are also applicable to other positionsof the tetracycline compound. Additional derivatizing agents andsecondary appropriate conditions may be found, for example, in thechemical literature. See, for example, R. C. LaRock, ComprehensiveOrganic Transformations, (New York: VCH Publishers, Inc., 1989) andreferences cited therein. Any reagent that can react with a primaryamine to form a new compound is possible. Examples of some of thediverse structures are shown in Scheme 4 below

For example, in Scheme 5, an acid chloride derivatizing agent is addedto the reaction mixture to form the desired amide aminoalkyltetracycline compound (J. Am. Chem. Soc. 71, 2215 (1949); J. Am. Chem.Soc. 108, 1039 (1986); Org. Syn. Coll. Vol. 4, 339 (1963); Org. Syn.Coll. Vol. 5, 387 (1973)).

Scheme 6 depicts the reaction of an intermediate aminoalkyl tetracyclinecompound with an appropriate sulfonyl chloride derivatizing agent, suchthat the desired sulfonamide aminoalkyl compound is formed (Org. Syn.Coll. Vol. 5, 736, 758 (1973)).

Scheme 7 depicts the reaction of a derivatizing agent with an aminoalkyltetracycline intermediate to form the resulting carbamate aminoalkyltetracycline compound.

The term “alkyl” includes saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. The term alkyl further includes alkyl groups,which can further include oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more carbons of the hydrocarbon backbone, e.g., oxygen,nitrogen, sulfur or phosphorous atoms. In preferred embodiments, astraight chain or branched chain alkyl has 6 or fewer carbon atoms inits backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ for branched chain),and more preferably 4 or fewer. Likewise, preferred cycloalkyls havefrom 4-7 carbon atoms in their ring structure, and more preferably have5 or 6 carbons in the ring structure.

Moreover, the term alkyl includes both “unsubstituted alkyls” and“substituted alkyls”, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, alkylthiocarbonyl, alkoxyl, aryloxy, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromaticmoiety. Cycloalkyls can be further substituted, e.g., with thesubstituents described above. An “alkylaryl” moiety is an alkylsubstituted with an aryl (e.g., phenylmethyl(benzyl)).

The term “aryl” includes aryl groups, including 5- and 6-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine,pyrazine, pyridazine and pyrimidine, and the like. Aryl groups alsoinclude polycyclic fused aromatic groups such as naphthyl, quinolyl,indolyl, and the like. Those aryl groups having heteroatoms in the ringstructure may also be referred to as “aryl heterocycles”, “heteroaryls”or “heteroaromatics”. The aromatic ring can be substituted at one ormore ring positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromaticmoiety. Aryl groups can also be fused or bridged with alicyclic orheterocyclic rings which are not aromatic so as to form a polycycle(e.g., tetralin).

The terms “alkenyl” and “alkynyl” include unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto five carbon atoms in its backbone structure. Likewise, “loweralkenyl” and “lower alkynyl” have similar chain lengths.

The terms “alkoxyalkyl”, “polyaminoalkyl” and “thioalkoxyalkyl” includealkyl groups, as described above, which further include oxygen, nitrogenor sulfur atoms replacing one or more carbons of the hydrocarbonbackbone, e.g., oxygen, nitrogen or sulfur atoms.

The terms “polycyclyl” or “polycyclic radical” refer to two or morecyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, arylsand/or heterocyclyls) in which two or more carbons are common to twoadjoining rings, e.g., the rings are “fused rings”. Rings that arejoined through non-adjacent atoms are termed “bridged” rings. Each ofthe rings of the polycycle can be substituted with such substituents asdescribed above, as for example, halogen, hydroxyl, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (includingalkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or anaromatic or heteroaromatic moiety.

The term “heterocyclic ring” refers to a ring containing one or moreheteroatoms as part of the ring. Examples of “heterocyclic rings”include tetrahydrofuran, furan, ethylene oxide, pyrrolidine, piperidine,thiophene, and pyrrole.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,sulfur and phosphorus.

The term “bicyclic ring” includes rings where 2 atoms share more thanone ring. Examples of bicyclic rings include bicyclbutane, camphene,decalin, and phthalimide.

The term “carbonyl” includes moieties which contain a carbon doublebonded to an oxygen atom. The term “substituted carbonyl” includesgroups wherein the carbon of the carbonyl group is further bonded toanother carbon or a heteroatom. Examples of substituents include, butare not limited to, alkyl, alkenyl, alkynyl, aryl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, alkoxy, aryloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,aphosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

The term “thiocarbonyl” includes moieties which contain a carbon doublebonded to a sulfur atom. The term “substituted thiocarbonyl” includesgroups wherein the carbon of the carbonyl group is further bonded toanother carbon or a heteroatom. Examples of substituents include, butare not limited to, alkyl, alkenyl, alkynyl, aryl, halogen, hydroxyl,alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, alkoxy, aryloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,aphosphate, phosphonato, phosphinato, cyano, amino (including alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

Suitable alkanoyl groups include groups having 1 to about 4 or 5carbonyl groups. Suitable aroyl groups include groups having one or morecarbonyl groups as a substituent to an aryl group such as phenyl orother carbocyclic aryl. Suitable alkaroyl groups have one or morealkylcarbonyl groups as a substituent to an aryl group such asphenylacetyl and the like. Suitable carbocyclic aryl groups have 6 ormore carbons such as phenyl, naphthyl and the like. Suitable aryloylgroups are carbocyclic aryl groups that are substituted with one or morecarbonyl groups, typically 1 or 2 carbonyl groups.

Prodrugs are compounds which are converted in vivo to active forms (see,e.g., R. B. Silverman, 1992, “The Organic Chemistry of Drug Design andDrug Action”, Academic Press, Chp. 8). Prodrugs can be used to alter thebiodistribution (e.g., to allow compounds which would not typicallyenter the reactive site of the protease) or the pharmacokinetics for aparticular compound. For example, a hydroxyl group, can be esterified,e.g., with a carboxylic acid group to yield an ester. When the ester isadministered to a subject, the ester is cleaved, enzymatically ornon-enzymatically, reductively or hydrolytically, to reveal the hydroxylgroup. Prodrugs may be metabolized in vivo by esterases or by othermechanisms to hydroxyl groups or other advantageous groups. Examples ofprodrugs and their uses are well known in the art (See, e.g., Berge etal. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). The prodrugscan be prepared in situ during the final isolation and purification ofthe compounds, or by separately reacting the purified compound in itsfree acid form or hydroxyl with a suitable esterifying agent. Hydroxylgroups can be converted into esters via treatment with a carboxylicacid. Examples of prodrugs include substituted and unsubstituted, branchor unbranched lower alkyl ester moieties, (e.g., propionoic acidesters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters(e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g.,acetyloxymethyl ester), acyloxy lower alkyl esters (e.g.,pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkylesters (e.g., benzyl ester), substituted (e.g., with methyl, halo, ormethoxy substituents) aryl and aryl-lower alkyl esters, amides,lower-alkyl amides, di-lower alkyl amides, and hydroxy amides.

It will be noted that the structure of some of the compounds of thisinvention includes asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thisinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis.

The invention also pertains, at least in part, to methods of treating asubject, e.g., a mammal, e.g., a human, for a tetracycline responsivestate by administering an effective amount of an aminomethyltetracycline compound of the invention, e.g., a compound of Formula (I),(III), (III), Table 1, or otherwise described herein.

The language “tetracycline compound responsive state” or “tetracyclineresponsive state” includes states which can be treated, prevented, orotherwise ameliorated by the administration of a tetracycline compoundof the invention, e.g., a 9-aminomethyl tetracycline compound.Tetracycline compound responsive states include bacterial, viral, andfungal infections (including those which are resistant to othertetracycline compounds), cancer (e.g., prostate, breast, colon, lungmelanoma and lymph cancers and other disorders characterized by unwantedcellular proliferation, including, but not limited to, those describedin U.S. Pat. No. 6,100,248), arthritis, osteoporosis, diabetes, cysticfibrosis, neurological disorders and other states for which tetracyclinecompounds have been found to be active (see, for example, U.S. Pat. Nos.5,789,395; 5,834,450; 6,277,061 and 5,532,227, each of which isexpressly incorporated herein by reference). Compounds of the inventioncan be used to prevent or control important mammalian and veterinarydiseases such as diarrhea, urinary tract infections, infections of skinand skin structure, ear, nose and throat infections, wound infection,mastitis and the like. In addition, methods for treating neoplasms usingtetracycline compounds of the invention are also included (van derBozert et al., Cancer Res., 48:6686-6690 (1988)). In a furtherembodiment, the tetracycline responsive state is not a bacterialinfection. Other tetracycline compound responsive states include, forexample, those described in U.S. Ser. No. 10/196,010.

Tetracycline compound responsive states also include inflammatoryprocess associated states (IPAS). The term “inflammatory processassociated state” includes states in which inflammation or inflammatoryfactors (e.g., matrix metalloproteinases (MMPs), nitric oxide (NO), TNF,interleukins, plasma proteins, cellular defense systems, cytokines,lipid metabolites, proteases, toxic radicals, adhesion molecules, etc.)are involved or are present in an area in aberrant amounts, e.g., inamounts which may be advantageous to alter, e.g., to benefit thesubject. The inflammatory process is the response of living tissue todamage. The cause of inflammation may be due to physical damage,chemical substances, micro-organisms, tissue necrosis, cancer or otheragents. Acute inflammation is short-lasting, lasting only a few days. Ifit is longer lasting however, then it may be referred to as chronicinflammation.

IPAS's include inflammatory disorders. Inflammatory disorders aregenerally characterized by heat, redness, swelling, pain and loss offunction. Examples of causes of inflammatory disorders include, but arenot limited to, microbial infections (e.g., bacterial and fungalinfections), physical agents (e.g., burns, radiation, and trauma),chemical agents (e.g., toxins and caustic substances), tissue necrosisand various types of immunologic reactions.

Examples of inflammatory disorders include, but are not limited to,osteoarthritis, rheumatoid arthritis, acute and chronic infections(bacterial and fungal, including diphtheria and pertussis); acute andchronic bronchitis, sinusitis, and upper respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns (thermal, chemical, and electrical); and sunburn.

Tetracycline compound responsive states also include NO associatedstates. The term “NO associated state” includes states which involve orare associated with nitric oxide (NO) or inducible nitric oxide synthase(iNOS). NO associated state includes states which are characterized byaberrant amounts of NO and/or iNOS. Preferably, the NO associated statecan be treated by administering tetracycline compounds of the invention,e.g., compounds of formula I, II, III, Table 1, or otherwise describedherein. The disorders, diseases and states described in U.S. Pat. Nos.6,231,894; 6,015,804; 5,919,774; and 5,789,395 are also included as NOassociated states. The entire contents of each of these patents arehereby incorporated herein by reference.

Other examples of NO associated states include, but are not limited to,malaria, senescence, diabetes, vascular stroke, neurodegenerativedisorders (Alzheimer's disease, Huntington's disease), cardiac disease(reperfusion-associated injury following infarction), juvenile diabetes,inflammatory disorders, osteoarthritis, rheumatoid arthritis, acute andchronic infections (bacterial, viral, and fungal); cystic fibrosis,acute and chronic bronchitis, sinusitis, and respiratory infections,including the common cold; acute and chronic gastroenteritis andcolitis; acute and chronic cystitis and urethritis; acute and chronicdermatitis; acute and chronic conjunctivitis; acute and chronicserositis (pericarditis, peritonitis, synovitis, pleuritis andtendinitis); uremic pericarditis; acute and chronic cholecystis; acuteand chronic vaginitis; acute and chronic uveitis; drug reactions; insectbites; burns (thermal, chemical, and electrical); and sunburn.

The term “inflammatory process associated state” also includes, in oneembodiment, matrix metalloproteinase associated states (MMPAS). MMPASinclude states characterized by aberrant amounts of MMPs or MMPactivity. These are also include as tetracycline compound responsivestates which may be treated using compounds of the invention, e.g.,9-aminomethyl tetracycline compounds such as those described herein,e.g., in formula I.

Examples of matrix metalloproteinase associated states (“MMPAS's”)include, but are not limited to, arteriosclerosis, corneal ulceration,emphysema, osteoarthritis, multiple sclerosis (Liedtke et al., Ann.Neurol. 1998, 44:35-46; Chandler et al., J. Neuroimmunol. 1997,72:155-71), osteosarcoma, osteomyelitis, bronchiectasis, chronicpulmonary obstructive disease, skin and eye diseases, periodontitis,osteoporosis, rheumatoid arthritis, ulcerative colitis, inflammatorydisorders, tumor growth and invasion (Stetler-Stevenson et al., Annu.Rev. Cell Biol. 1993, 9:541-73; Tryggvason et al., Biochim. Biophys.Acta 1987, 907:191-217; Li et al., Mol. Carcinog. 1998, 22:84-89),metastasis, acute lung injury, stroke, ischemia, diabetes, aortic orvascular aneurysms, skin tissue wounds, dry eye, bone and cartilagedegradation (Greenwald et al., Bone 1998, 22:33-38; Ryan et al., Curr.Op. Rheumatol. 1996, 8; 238-247). Other MMPAS include those described inU.S. Pat. Nos. 5,459,135; 5,321,017; 5,308,839; 5,258,371; 4,935,412;4,704,383, 4,666,897, and RE 34,656, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state iscancer. Examples of cancers which the tetracycline compounds of theinvention may be useful to treat include all solid tumors, i.e.,carcinomas e.g., adenocarcinomas, and sarcomas. Adenocarcinomas arecarcinomas derived from glandular tissue or in which the tumor cellsform recognizable glandular structures. Sarcomas broadly include tumorswhose cells are embedded in a fibrillar or homogeneous substance likeembryonic connective tissue. Examples of carcinomas which may be treatedusing the methods of the invention include, but are not limited to,carcinomas of the prostate, breast, ovary, testis, lung, colon, andbreast. The methods of the invention are not limited to the treatment ofthese tumor types, but extend to any solid tumor derived from any organsystem. Examples of treatable cancers include, but are not limited to,colon cancer, bladder cancer, breast cancer, melanoma, ovariancarcinoma, prostatic carcinoma, lung cancer, and a variety of othercancers as well. The methods of the invention also cause the inhibitionof cancer growth in adenocarcinomas, such as, for example, those of theprostate, breast, kidney, ovary, testes, and colon.

In an embodiment, the tetracycline responsive state of the invention iscancer. The invention pertains to a method for treating a subjectsuffering or at risk of suffering from cancer, by administering aneffective amount of a substituted tetracycline compound, such thatinhibition cancer cell growth occurs, i.e., cellular proliferation,invasiveness, metastasis, or tumor incidence is decreased, slowed, orstopped. The inhibition may result from inhibition of an inflammatoryprocess, down-regulation of an inflammatory process, some othermechanism, or a combination of mechanisms. Alternatively, thetetracycline compounds may be useful for preventing cancer recurrence,for example, to treat residual cancer following surgical resection orradiation therapy. The tetracycline compounds useful according to theinvention are especially advantageous as they are substantiallynon-toxic compared to other cancer treatments. In a further embodiment,the compounds of the invention are administered in combination withstandard cancer therapy, such as, but not limited to, chemotherapy.

The language “in combination with” another therapeutic agent ortreatment includes co-administration of the tetracycline compound andwith the other therapeutic agent or treatment, administration of thetetracycline compound first, followed by the other therapeutic agent ortreatment and administration of the other therapeutic agent or treatmentfirst, followed by the tetracycline compound. The other therapeuticagent may be any agent which is known in the art to treat, prevent, orreduce the symptoms of a tetracycline responsive state. Furthermore, theother therapeutic agent may be any agent of benefit to the patient whenadministered in combination with the administration of an tetracyclinecompound. In one embodiment, the cancers treated by methods of theinvention include those described in U.S. Pat. Nos. 6,100,248;5,843,925; 5,837,696; or 5,668,122, incorporated herein by reference intheir entirety.

In another embodiment, the tetracycline compound responsive state isdiabetes, e.g., juvenile diabetes, diabetes mellitus, diabetes type I,diabetes type II, diabetic ulcers, or other diabetic complications. In afurther embodiment, protein glycosylation is not affected by theadministration of the tetracycline compounds of the invention. Inanother embodiment, the tetracycline compound of the invention isadministered in combination with standard diabetic therapies, such as,but not limited to insulin therapy. In a further embodiment, the IPASincludes disorders described in U.S. Pat. Nos. 5,929,055; and 5,532,227,incorporated herein by reference in their entirety.

In another embodiment, the tetracycline compound responsive state is abone mass disorder. Bone mass disorders include disorders where asubjects bones are disorders and states where the formation, repair orremodeling of bone is advantageous. For examples bone mass disordersinclude osteoporosis (e.g., a decrease in bone strength and density),bone fractures, bone formation associated with surgical procedures(e.g., facial reconstruction), osteogenesis imperfecta (brittle bonedisease), hypophosphatasia, Paget's disease, fibrous dysplasia,osteopetrosis, myeloma bone disease, and the depletion of calcium inbone, such as that which is related to primary hyperparathyroidism. Bonemass disorders include all states in which the formation, repair orremodeling of bone is advantageous to the subject as well as all otherdisorders associated with the bones or skeletal system of a subjectwhich can be treated with the tetracycline compounds of the invention.In a further embodiment, the bone mass disorders include those describedin U.S. Pat. Nos. 5,459,135; 5,231,017; 5,998,390; 5,770,588; RE 34,656;5,308,839; 4,925,833; 3,304,227; and 4,666,897, each of which is herebyincorporated herein by reference in its entirety.

In another embodiment, the tetracycline compound responsive state isacute lung injury. Acute lung injuries include adult respiratorydistress syndrome (ARDS), post-pump syndrome (PPS), and trauma. Traumaincludes any injury to living tissue caused by an extrinsic agent orevent. Examples of trauma include, but are not limited to, crushinjuries, contact with a hard surface, or cutting or other damage to thelungs.

The invention also pertains to a method for treating acute lung injuryby administering a tetracycline compound of the invention.

The tetracycline responsive states of the invention also include chroniclung disorders. The invention pertains to methods for treating chroniclung disorders by administering a tetracycline compound, such as thosedescribed herein. The method includes administering to a subject aneffective amount of a substituted tetracycline compound such that thechronic lung disorder is treated. Examples of chronic lung disordersinclude, but are not limited, to asthma, cystic fibrosis, and emphysema.In a further embodiment, the tetracycline compounds of the inventionused to treat acute and/or chronic lung disorders such as thosedescribed in U.S. Pat. Nos. 5,977,091; 6,043,231; 5,523,297; and5,773,430, each of which is hereby incorporated herein by reference inits entirety.

In yet another embodiment, the tetracycline compound responsive state isischemia, stroke, or ischemic stroke. The invention also pertains to amethod for treating ischemia, stroke, or ischemic stroke byadministering an effective amount of a substituted tetracycline compoundof the invention. In a further embodiment, the tetracycline compounds ofthe invention are used to treat such disorders as described in U.S. Pat.No. 6,231,894; 5,773,430; 5,919,775 or 5,789,395, incorporated herein byreference.

In another embodiment, the tetracycline compound responsive state is askin wound. The invention also pertains, at least in part, to a methodfor improving the healing response of the epithelialized tissue (e.g.,skin, mucosae) to acute traumatic injury (e.g., cut, burn, scrape,etc.). The method may include using a tetracycline compound of theinvention (which may or may not have antibacterial activity) to improvethe capacity of the epithelialized tissue to heal acute wounds. Themethod may increase the rate of collagen accumulation of the healingtissue. The method may also decrease the proteolytic activity in theepthithelialized tissue by decreasing the collagenolytic and/orgelatinolytic activity of MMPs. In a further embodiment, thetetracycline compound of the invention is administered to the surface ofthe skin (e.g., topically). In a further embodiment, the tetracyclinecompound of the invention is used to treat a skin wound, and other suchdisorders as described in, for example, U.S. Pat. Nos. 5,827,840;4,704,383; 4,935,412; 5,258,371; 5,308,8391 5,459,135; 5,532,227; and6,015,804; each of which is incorporated herein by reference in itsentirety.

Examples of tetracycline responsive states also include neurologicaldisorders which include both neuropsychiatric and neurodegenerativedisorders, but are not limited to, such as Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, senile dementia,Huntington's disease, Gilles de la Tourette's syndrome, multiplesclerosis, amyotrophic lateral sclerosis (ALS), progressive supranuclearpalsy, epilepsy, and Creutzfeldt-Jakob disease; autonomic functiondisorders such as hypertension and sleep disorders, and neuropsychiatricdisorders, such as depression, schizophrenia, schizoaffective disorder,Korsakoff's psychosis, mania, anxiety disorders, or phobic disorders;learning or memory disorders, e.g., amnesia or age-related memory loss,attention deficit disorder, dysthymic disorder, major depressivedisorder, mania, obsessive-compulsive disorder, psychoactive substanceuse disorders, anxiety, phobias, panic disorder, as well as bipolaraffective disorder, e.g., severe bipolar affective (mood) disorder(BP-1), bipolar affective neurological disorders, e.g., migraine andobesity. Further neurological disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.

In yet another embodiment, the tetracycline compound responsive state isan aortic or vascular aneurysm in vascular tissue of a subject (e.g., asubject having or at risk of having an aortic or vascular aneurysm,etc.). The tetracycline compound may by effective to reduce the size ofthe vascular aneurysm or it may be administered to the subject prior tothe onset of the vascular aneurysm such that the aneurysm is prevented.In one embodiment, the vascular tissue is an artery, e.g., the aorta,e.g., the abdominal aorta. In a further embodiment, the tetracyclinecompounds of the invention are used to treat disorders described in U.S.Pat. Nos. 6,043,225 and 5,834,449, incorporated herein by reference intheir entirety.

Bacterial infections may be caused by a wide variety of gram positiveand gram negative bacteria. The compounds of the invention are useful asantibiotics against organisms which are resistant to other tetracyclinecompounds. The antibiotic activity of the tetracycline compounds of theinvention may be determined using the method discussed in Example 2, orby using the in vitro standard broth dilution method described in Waitz,J. A., National Commission for Clinical Laboratory Standards, DocumentM7-A2, vol. 10, no. 8, pp. 13-20, 2^(nd) edition, Villanova, Pa. (1990).

The tetracycline compounds of the invention may also be used to treatinfections traditionally treated with tetracycline compounds such as,for example, rickettsiae; a number of gram-positive and gram-negativebacteria; and the agents responsible for lymphogranuloma venereum,inclusion conjunctivitis, psittacosis. The tetracycline compounds may beused to treat infections of, e.g., K. pneumoniae, Salmonella, E. hirae,A. baumanii, B. catarrhalis, H. influenzae, P. aeruginosa, E. faecium,E. coli, S. aureus or E. faecalis. In one embodiment, the tetracyclinecompound is used to treat a bacterial infection that is resistant toother tetracycline antibiotic compounds. The tetracycline compound ofthe invention may be administered with a pharmaceutically acceptablecarrier.

The language “effective amount” of the compound is that amount necessaryor sufficient to treat or prevent a tetracycline compound responsivestate. The effective amount can vary depending on such factors as thesize and weight of the subject, the type of illness, or the particulartetracycline compound. For example, the choice of the tetracyclinecompound can affect what constitutes an “effective amount”. One ofordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thetetracycline compound without undue experimentation.

In an embodiment, the invention pertains to pharmaceutical compositioncomprising a tetracycline compound of the invention, e.g., a compound ofFormula (I), (II), (III), or otherwise described herein. Preferrably,the tetracycline compound is provided in an effective amount, e.g.,effective to treat a tetracycline responsive state in a subject, e.g., amammal, e.g., a human. In a further embodiment, the pharmaceuticalcomposition of the invention also comprises an appropriatepharmaceutically acceptable carrier.

The language “pharmaceutically acceptable carrier” includes substancescapable of being coadministered with the tetracycline compound(s), andwhich allow both to perform their intended function, e.g., treat orprevent a tetracycline compound responsive state. Suitablepharmaceutically acceptable carriers include but are not limited towater, salt solutions, alcohol, vegetable oils, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds of the invention.

The tetracycline compounds of the invention that are basic in nature arecapable of forming a wide variety of salts with various inorganic andorganic acids. The acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of the tetracycline compounds of theinvention that are basic in nature are those that form non-toxic acidaddition salts, i.e., salts containing pharmaceutically acceptableanions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,lactate, salicylate, citrate, acid citrate, tartrate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand palmoate [i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)] salts.Although such salts must be pharmaceutically acceptable foradministration to a subject, e.g., a mammal, it is often desirable inpractice to initially isolate a tetracycline compound of the inventionfrom the reaction mixture as a pharmaceutically unacceptable salt andthen simply convert the latter back to the free base compound bytreatment with an alkaline reagent and subsequently convert the latterfree base to a pharmaceutically acceptable acid addition salt. The acidaddition salts of the base compounds of this invention are readilyprepared by treating the base compound with a substantially equivalentamount of the chosen mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon careful evaporation of the solvent, the desired solid salt isreadily obtained.

The tetracycline compounds of the invention that are acidic in natureare capable of forming a wide variety of base salts. The chemical basesthat may be used as reagents to prepare pharmaceutically acceptable basesalts of those tetracycline compounds of the invention that are acidicin nature are those that form non-toxic base salts with such compounds.Such non-toxic base salts include, but are not limited to those derivedfrom such pharmaceutically acceptable cations such as alkali metalcations (e.g., potassium and sodium) and alkaline earth metal cations(e.g., calcium and magnesium), ammonium or water-soluble amine additionsalts such as N-methylglucamine-(meglumine), and the loweralkanolammonium and other base salts of pharmaceutically acceptableorganic amines. The pharmaceutically acceptable base addition salts oftetracycline compounds of the invention that are acidic in nature may beformed with pharmaceutically acceptable cations by conventional methods.Thus, these salts may be readily prepared by treating the tetracyclinecompound of the invention with an aqueous solution of the desiredpharmaceutically acceptable cation and evaporating the resultingsolution to dryness, preferably under reduced pressure. Alternatively, alower alkyl alcohol solution of the tetracycline compound of theinvention may be mixed with an alkoxide of the desired metal and thesolution subsequently evaporated to dryness.

The preparation of other tetracycline compounds of the invention notspecifically described in the foregoing experimental section can beaccomplished using combinations of the reactions described above thatwill be apparent to those skilled in the art.

The tetracycline compounds of the invention and pharmaceuticallyacceptable salts thereof can be administered via either the oral,parenteral or topical routes. In general, these compounds are mostdesirably administered in effective dosages, depending upon the weightand condition of the subject being treated and the particular route ofadministration chosen. Variations may occur depending upon the speciesof the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

The pharmaceutical compositions of the invention may be administeredalone or in combination with other known compositions for treatingtetracycline responsive states in subjects, e.g., mammals. The subjectsmay be suffering from or at risk of suffering from a tetracyclineresponsive state. Examples of subjects include pets (e.g., cats, dogs,ferrets, etc.), farm animals (cows, sheep, pigs, horses, goats, etc.),lab animals (rats, mice, monkeys, etc.), and primates (chimpanzees,humans, gorillas).

The language “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the inventionand the known composition, administration of the composition of theinvention first, followed by the known composition and administration ofthe known composition first, followed by the composition of theinvention. Any of the therapeutically composition known in the art fortreating tetracycline responsive states can be used in the methods ofthe invention.

The compounds of the invention may be administered alone or incombination with pharmaceutically acceptable carriers or diluents by anyof the routes previously mentioned, and the administration may becarried out in single or multiple doses. For example, the noveltherapeutic agents of this invention can be administered advantageouslyin a wide variety of different dosage forms, i.e., they may be combinedwith various pharmaceutically acceptable inert carriers in the form oftablets, capsules, lozenges, troches, hard candies, powders, sprays,creams, salves, suppositories, jellies, gels, pastes, lotions,ointments, aqueous suspensions, injectable solutions, elixirs, syrups,and the like. Such carriers include solid diluents or fillers, sterileaqueous media and various non-toxic organic solvents, etc. Moreover,oral pharmaceutical compositions can be suitably sweetened and/orflavored. In general, the therapeutically-effective compounds of thisinvention are present in such dosage forms at concentration levelsranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

For parenteral administration (including intraperitoneal, subcutaneous,intravenous, intradermal or intramuscular injection), solutions of atherapeutic compound of the present invention in either sesame or peanutoil or in aqueous propylene glycol may be employed. The aqueoussolutions should be suitably buffered (preferably pH greater than 8) ifnecessary and the liquid diluent first rendered isotonic. These aqueoussolutions are suitable for intravenous injection purposes. The oilysolutions are suitable for intraarticular, intramuscular andsubcutaneous injection purposes. The preparation of all these solutionsunder sterile conditions is readily accomplished by standardpharmaceutical techniques well known to those skilled in the art. Forparenteral application, examples of suitable preparations includesolutions, preferably oily or aqueous solutions as well as suspensions,emulsions, or implants, including suppositories. Therapeutic compoundsmay be formulated in sterile form in multiple or single dose formatssuch as being dispersed in a fluid carrier such as sterile physiologicalsaline or 5% saline dextrose solutions commonly used with injectables.

Additionally, it is also possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin. Examples of methods of topical administration include transdermal,buccal or sublingual application. For topical applications, therapeuticcompounds can be suitably admixed in a pharmacologically inert topicalcarrier such as a gel, an ointment, a lotion or a cream. Such topicalcarriers include water, glycerol, alcohol, propylene glycol, fattyalcohols, triglycerides, fatty acid esters, or mineral oils. Otherpossible topical carriers are liquid petrolatum, isopropylpalmitate,polyethylene glycol, ethanol 95%, polyoxyethylene monolauriate 5% inwater, sodium lauryl sulfate 5% in water, and the like. In addition,materials such as anti-oxidants, humectants, viscosity stabilizers andthe like also may be added if desired.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

In addition to treatment of human subjects, the therapeutic methods ofthe invention also will have significant veterinary applications, e.g.for treatment of livestock such as cattle, sheep, goats, cows, swine andthe like; poultry such as chickens, ducks, geese, turkeys and the like;horses; and pets such as dogs and cats. Also, the compounds of theinvention may be used to treat non-animal subjects, such as plants.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of administration can bereadily ascertained by those skilled in the art using conventionaldosage determination tests conducted with regard to the foregoingguidelines.

In general, compounds of the invention for treatment may, in certainembodiments, be administered to a subject in dosages used in priortetracycline therapies. See, for example, the Physicians' DeskReference. For example, a suitable effective dose of one or morecompounds of the invention will be in the range of from 0.01 to 100milligrams per kilogram of body weight of recipient per day, preferablyin the range of from 0.1 to 50 milligrams per kilogram body weight ofrecipient per day, more preferably in the range of 1 to 20 milligramsper kilogram body weight of recipient per day. The desired dose issuitably administered once daily, or several sub-doses, e.g. 2 to 5sub-doses, are administered at appropriate intervals through the day, orother appropriate schedule.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of tetracyclines generally toensure their efficacy under normal use circumstances. Especially whenemployed for therapeutic treatment of humans and animals in vivo, thepractitioner should take all sensible precautions to avoidconventionally known contradictions and toxic effects. Thus, theconventionally recognized adverse reactions of gastrointestinal distressand inflammations, the renal toxicity, hypersensitivity reactions,changes in blood, and impairment of absorption through aluminum,calcium, and magnesium ions should be duly considered in theconventional manner.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXEMPLIFICATION OF THE INVENTION Example 1 Synthesis of 9-AminomethylMinocycline and Derivatives Thereof

Trifluoroacetic acid (1 L) was charged into a 2 L flask under argon andminocycline. HCl (200 g, 1 eq) and N-hydroxymethylphthalimide (100 g)were added to the flask while stirring. Once the entire solid dissolved,H₂SO₄ (200 mL) was added to the reaction. The reaction was heated to40-50° C. for 5-6 hours. N-hydroxymethylamine (10 g) was addedportionwise. When HPLC analysis confirmed that all the starting materialwas converted to 2,9-bis-aminomethylphthalimidominocycline, the mixturewas precipitated out of 4 L of acetone. An exotherm of 15-20° C. wasobserved. After 1 hour of stirring, the solid was filtered, washed withacetone (200 ml), and dried with the aid of a latex rubber dam. Thesolid was reslurried in a methanol (1 L)/t-BME (2 L) mixture and the pHwas adjusted to 3 using triethylamine. The solid was filtered and washedwith 50 mL of methanol. The yield was 97% of2,9-bis-aminomethylphthalimideminocycline.

2,9-bis-aminomethylphthalimideminocycline (100 g) was suspended in 2Msolution of methylamine in methanol (10 eq). The reaction was stirred atroom temperature for 2-3 hours, at which point HPLC analysis confirmedtotal conversion of the starting material to 2,9-bisaminomethylminocycline. The reaction mixture was poured into t-BME (5volumes), and stirred for thirty minutes. Next, the suspension wasfiltered and washed with t-BME (200 mL) to isolate the desired product,2,9-bis-aminomethylminocycline.

2,9-bis-aminomethylminocycline (40 g) was slurried in 200 mLwater/methanol 1/9 and the pH was adjusted to 3 by the dropwise additionof trifluoroacetic acid. The mixture was heated to 40° C. for 1-2 hours.When HPLC analysis confirmed the hydrolysis of2,9-bis-aminomethylminocycline to 9-aminomethylminocycline, the reactionwas allowed to return to room temperature and the pH was adjusted to 7using triethylamine. Isopropyl alcohol (200 mL) was added to precipitateout the solid. The product was filtered and washed with 50 mL IPAfollowed by 100 mL diethyl ether and dried under reduced pressuretoisolate 9-aminomethylminocycline.

9-[(Benzylamino)-methyl]-minocycline dihydrochloride

To 1.0 mmol (600 mg) of 9-(aminomethyl)-minocycline dihydrochloride andin 5 mL of dimethylformamide was added 0.2 mmol (5 mg) of indiumtrichloride and 1.5 mmol (160 mg) of benzaldehyde at room temperature.After 30 minutes of shaking, 2 mmol (424 mg) of sodiumtriacetoxyborohydride was added and the reaction was monitored by HPLC.After 1.5 hours, 3 equivalents of triethylamine and 1 equivalent ofsodium triacetoxyborohydride. The reaction was complete after 3 hours.The solvent was removed in vacuo and the crude product was purified bypreparative HPLC to yield 60 mg of 9-[(benzylamino)-methyl]-minocyclinedihydrochloride. LCMS (MH+)=577.

9-[(2,2, dimethyl-propyl amino)-methyl]-minocycline dihydrochloride

9-dimethylaminominocycline (200 mg, 1 eq.), DMF, andtrimethylacetaldehyde (45 μl, 1 eq.) were combined in 40 mL flasks andstirred. Triethylamine (150 μL, 3 eq.) was then added. After stirring atroom temperature for several minutes, NaBH(OAc)₃ (175 mg, 2 eq.) andInCl₃ (9 mg, 0.1 eq.) was added. After one hour, the reactions wereclear and red. Liquid chromatography showed a single product for thereaction. The reaction was quenched with methanol, the solvent wasremoved, and the product was purified using column chromatography.

9-[3,4-(Methylenedioxo)phenyl-ureido]-methylminocycline dihydrochloride

To 0.25 mmol (150 mg) of 9-(aminomethyl)-minocycline dihydrochloride and2 equivalents of triethylamine in 3 mL of dimethylformamide was added0.5 mmol (81.5 mg) of 3,4-(methylenedioxo)phenyl isocyanate at roomtemperature. Solution was shaken until reaction was complete (3 hours).Solvent was removed in vacuo and crude product was purified bypreparative HPLC to yield 66 mg of9-[3,4-(methylenedioxo)phenyl-ureido]-methylminocycline dihydrochloride.Yield 41%. LCMS (MH+)=650.

9-[4-(Trifluoromethoxy)phenyl-ureido]-methylminocycline dihydrochloride

To 0.25 mmol (150 mg) of 9-(aminomethyl)-minocycline dihydrochloride and2 equivalents of triethylamine in 3 mL of dimethylformamide was added0.5 mmol (101.5 mg) of 4-(trifluoromethoxy)phenyl isocyanate at roomtemperature. The solution was shaken until the reaction was complete (3hours). Solvent was removed in vacuo and crude product was purified bypreparative HPLC to yield 68 mg of9-[4-(trifluoromethoxy)phenyl-ureido]-methylminocycline dihydrochloride.Yield 39%. LCMS (MH+)=690.

7-[(Bis-dimethoxymethyl-amino)-methyl]-sancycline dihydrochloride

To 1.34 mmol (1 g) of 7-(aminomethyl)-sancycline dihydrochloride and 2equivalents of triethylamine in 5 mL of dimethylformamide was added0.134 mmol (29 mg) of indium trichloride and 2.68 mmol (465 mg) of 60%aqueous dimethoxyacetaldehyde at room temperature. After 30 minutes ofshaking, 2.68 mmol (568 mg) of sodium triacetoxyborohydride was addedand the reaction was monitored by HPLC. The reaction was complete after1 hour. The solvent was removed in vacuo and the crude product waspurified by preparative HPLC to yield 100 mg of7-[(Bis-dimethoxymethyl-amino)-methyl]-sancycline dihydrochloride. LCMS(MH+)=620.

9-(2′-Phenyl-ethyl-1′-amino)-methyl]-doxycycline

Under an N₂ atmosphere, a stirred solution of 9-aminomethyldoxycyclinedihydrochloride (1.21 g, 2.21 mmol) in DMF (10 mL), was treated withInCl₃ (0.076 g, 0.34 mmol) and phenylacetaldehyde (0.511 mL; 4.4 mmol).HPLC and LCMS monitoring of the reaction indicated the completeconsumption of the starting material over the course of 12 hours. Theproducts were both the mono-(major) and bis-(minor) substitutedaminodoxycyclines. Methanol (10 mL) was added to quench the reaction.The reaction mixture was filtered through a bed of Celite, the celitewashed with methanol (2×5 mL), and the combined organic layer wasconcentrated to about 7-8 mL and diluted with ether. The resultingamorphous solid was filtered, washed with ether (6×15 mL) and driedunder vacuum to afford a red powder, which was purified by preparativeHPLC. The final product was characterized by HPLC, MS, and ¹H NMRspectroscopic methods. MS (m/z): Theor. 577.24; Found: 578.17 (M+1).

Example 2 In Vitro Minimum Inhibitory Concentration (MIC) Assay

The following assay is used to determine the efficacy of tetracyclinederivatives compounds against common bacteria. 2 mg of each compound isdissolved in 100 μl of DMSO. The solution is then added tocation-adjusted Mueller Hinton broth (CAMHB), which results in a finalcompound concentration of 200 μg per ml. The compound solutions arediluted to 50 μL volumes, with a test compound concentration of 0.098μg/ml. Optical density (OD) determinations are made from fresh log-phasebroth cultures of the test strains. Dilutions are made to achieve afinal cell density of 1×10⁶ CFU/ml. At OD=1, cell densities fordifferent genera should be approximately: E. coli 1 × 10⁹ CFU/ml S.aureus 5 × 10⁸ CFU/ml Enterococcus sp. 2.5 × 10⁹ CFU/ml

50 μl of the cell suspensions are added to each well of microtiterplates. The final cell density should be approximately 5×10⁵ CFU/ml.These plates are incubated at 35° C. in an ambient air incubator forapproximately 18 hr. The plates are read with a microplate reader andare visually inspected when necessary. The MIC is defined as the lowestconcentration of the compound that inhibits growth.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

The application is related to “9-Substituted Minocycline Compounds,” WO02/04406, filed Jun. 29, 2001, the entire contents of which are herebyincorporated herein by reference.

1. A tetracycline compound of the formula (I):

wherein R¹ and R² are linked to form a ring, or pharmaceuticallyacceptable salts, prodrugs and esters thereof.
 2. The tetracyclinecompound of claim 1, wherein R¹ and R² are linked to form a fivemembered ring.
 3. The tetracycline compound of claim 1, wherein R¹ andR² are linked to form a six membered ring.
 4. The tetracycline compoundof claim 3, wherein R¹ and R² are linked to form a piperidine ring,morpholine ring, pyridine ring, or a pyrazinyl ring.
 5. The tetracyclinecompound of claim 4, wherein said compound is:


6. A tetracycline compound of the formula:

pharmaceutically acceptable salts, esters or prodrugs thereof.
 7. Atetracycline compound of the formula (II):

wherein: J¹ and J² are each independently hydrogen, aryl, sulfonyl,acyl, or linked to form a ring, provided that at least one of J¹ or J²is not hydrogen; J³ and J⁴ are each alkyl, halogen, or hydrogen; X isCHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶, or O; R², R^(2′), R^(4′),and R^(4″) are each independently hydrogen, alkyl, alkenyl, alkynyl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl,aryl, heterocyclic, heteroaromatic or a prodrug moiety; R⁴ is NR^(4′),R^(4″), alkyl, alkenyl, alkynyl, aryl, hydroxyl, halogen, or hydrogen;R^(2′), R³, R¹⁰, R¹¹ and R¹² are each hydrogen or a pro-drug moiety; R⁵is hydroxyl, hydrogen, thiol, alkanoyl, aroyl, alkaroyl, aryl,heteroaromatic, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, arylalkyl, alkyl carbonyloxy,or aryl carbonyloxy; R⁶ and R^(6′) are each independently hydrogen,methylene, absent, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl,aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; R⁹ is hydrogen, nitro, alkyl, alkenyl, alkynyl, aryl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, arylalkyl, amino, arylalkenyl,arylalkynyl, thionitroso, or —(CH₂)₀₋₃NR^(9c)C(=Z′)ZR^(9a); Z isCR^(9d)R^(9e), NR^(9b) or O; Z′ is O, S, or NR^(9f); R^(9a), R^(9b),R^(9c), R^(9d), and R^(9e) are each independently hydrogen, acyl, alkyl,alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, arylalkyl, aryl, heterocyclic, heteroaromatic or a prodrugmoiety; R⁸ is hydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl,alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl,alkylamino, or an arylalkyl; R¹³ is hydrogen, hydroxy, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, oran arylalkyl; and Y′ and Y are each independently hydrogen, halogen,hydroxyl, cyano, sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl,and pharmaceutically acceptable salts, esters, and prodrugs thereof. 8.The tetracycline compound of claim 7, wherein R⁴ is NR^(4′)R^(4″), X isCR⁶R^(6′); R², R^(2′), R⁶, R^(6′), R⁸, R⁹, R¹⁰, R¹¹, and R¹² are eachhydrogen; R^(4′) and R^(4″) are lower alkyl; and R⁵ is hydroxy orhydrogen.
 9. The tetracycline compound of claim 8, wherein R^(4′) andR^(4″) are each methyl and R⁵ is hydrogen.
 10. The tetracycline compoundof claim 7, wherein J³ and J⁴ are hydrogen.
 11. The tetracyclinecompound of claim 7, wherein J¹ is substituted or unsubstituted alkyl.12. The tetracycline compound of claim 7, wherein J¹ is sulfonyl. 13.The tetracycline compound of claim 7, wherein J¹ and J² are linked toform a ring.
 14. The tetracycline compound of claim 7, wherein J¹ isheteroaryl.
 15. A tetracycline compound, wherein said compound isselected from the group consisting of:

wherein R is substituted or unsubstituted alkyl, alkenyl, alkynyl,halogen, alkoxy; and Y is N, O, or S, or pharmaceutically acceptablesalts, esters, or prodrugs thereof.
 16. A tetracycline compound offormula (III):

wherein: J⁵ and J⁶ are each independently hydrogen, alkyl, alkenyl,alkynyl, aryl, sulfonyl, acyl, alkoxycarbonyl, alkaminocarbonyl,alkaminothiocarbonyl, substituted thiocarbonyl, substituted carbonyl,alkoxythiocarbonyl, or linked to form a ring; J⁷ and J⁸ are each alkyl,halogen, or hydrogen; X is CHC(R¹³Y′Y), CR^(6′)R⁶, C═CR^(6′)R⁶, S, NR⁶,or O; R², R^(2′), R^(4′), and R^(4″) are each independently hydrogen,alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, arylalkyl, aryl, heterocyclic, heteroaromaticor a prodrug moiety; R⁴ is NR^(4′)R^(4″), alkyl, alkenyl, alkynyl, aryl,hydroxyl, halogen, or hydrogen; R^(2′), R³, R¹⁰, R¹¹ and R¹² are eachhydrogen or a pro-drug moiety; R⁵ is hydroxyl, hydrogen, thiol,alkanoyl, aroyl, alkaroyl, aryl, heteroaromatic, alkyl, alkenyl,alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino,arylalkyl, alkyl carbonyloxy, or aryl carbonyloxy; R⁶ and R^(6′) areeach independently hydrogen, methylene, absent, hydroxyl, halogen,thiol, alkyl, alkenyl, alkynyl, aryl, alkoxy, alkylthio, alkylsulfinyl,alkylsulfonyl, alkylamino, or an arylalkyl; R⁷ is hydrogen; R⁸ ishydrogen, hydroxyl, halogen, thiol, alkyl, alkenyl, alkynyl, aryl,alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or anarylalkyl; R¹³ is hydrogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy,alkylthio, alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl;and Y′ and Y are each independently hydrogen, halogen, hydroxyl, cyano,sulfhydryl, amino, alkyl, alkenyl, alkynyl, alkoxy, alkylthio,alkylsulfinyl, alkylsulfonyl, alkylamino, or an arylalkyl, andpharmaceutically acceptable salts thereof.
 17. The tetracycline compoundof claim 16, wherein R⁴ is NR^(4′)R^(4″), X is CR⁶R^(6′); R², R^(2′),R⁶, R^(6′), R⁸, R¹⁰, R¹¹, and R¹² are each hydrogen; R^(4′) and R^(4″)are lower alkyl; and R⁵ is hydroxy or hydrogen.
 18. The tetracyclinecompound of claim 17, wherein R^(4′) and R^(4″) are each methyl and R⁵is hydrogen.
 19. The tetracycline compound of claim 16, wherein J⁷ andJ⁸ are hydrogen.
 20. The tetracycline compound of claim 16, wherein J⁵is substituted or unsubstituted alkyl.
 21. The tetracycline compound ofclaim 16, wherein J⁵ is sulfonyl.
 22. The tetracycline compound of claim16, wherein J⁵ and J⁶ are linked to form a ring.
 23. The tetracyclinecompound of claim 16, wherein J⁵ is heteroaryl.
 24. The tetracyclinecompound of claim 16, wherein J⁵ is substituted carbonyl.
 25. Thetetracycline compound of claim 16, wherein said compound is selectedfrom the group consisting of:

wherein R is substituted or unsubstituted alkyl, alkenyl, alkynyl,halogen, alkoxy; and Y is N, O, or S, or pharmaceutically acceptablesalts or prodrugs thereof.
 26. A tetracycline compound of Table 1, or apharmaceutically acceptable salt thereof.
 27. A pharmaceuticalcomposition comprising an effective amount of a tetracycline compound ofany one of claims 1, 15, 16, 25 or 26, and a pharmaceutically acceptablecarrier.
 28. The pharmaceutical composition of claim 27, wherein saideffective amount is effective to treat a tetracycline responsive state.29. A method for treating a tetracycline responsive state in a subject,comprising administering to said subject a tetracycline compound of anyone of claims 1, 15, 16, 25 or 26, such that said subject is treated.30. The method of claim 29, wherein said tetracycline responsive stateis an inflammatory process associated state.
 31. The method of claim 29,wherein said tetracycline responsive state is cancer, a lung injury, aneye disorder, neurological disorder or stroke.
 32. The method of claim29, wherein said tetracycline responsive state is a bacterial infection.33. The method of claim 32, wherein said bacterial infection isassociated with E. coli.
 34. The method of claim 32, wherein saidbacterial infection is associated with S. aureus.
 35. The method ofclaim 32, wherein said bacterial infection is associated with E.faecalis.
 36. The method of claim 32, wherein said bacterial infectionis resistant to other tetracycline antibiotics.
 37. The method of claim32, wherein said bacterial infection is associated with gram positivebacteria.
 38. The method of claim 32, wherein said bacterial infectionis associated with gram negative bacteria.
 39. The method of claim 29,wherein said tetracycline responsive state is a viral or fungalinfection.
 40. The method of claim 29, wherein said tetracyclinecompound is administered with a pharmaceutically acceptable carrier. 41.The method of claim 29, wherein said subject is a human.